With the challenge of trying to pin down specific areas where microscopy developers could make significant strides in 2013, the editors at BioTechniques needed to huddle together and discussing (as well as debate) our various predictions.

Microscopy is an ever changing field, with new and significant advances
introduced on a regular basis. When it comes to areas such as fluorescent
proteins, resolution, data processing, automation, and unique applications
for these techniques, there is no doubt that 2013 will continue to bring
advances in each of these areas.

With the challenge of trying to pin down specific areas where microscopy developers could make significant strides in 2013, the editors at BioTechniques needed to huddle together and discussing (as well as debate) our various predictions. Source: RIKEN

The BioTechniques editors look forward to many changes in this field,
among them new technologies and refinement of existing approaches to close
the gap between light and electron microscopy capabilities. Methods for
sample preparation and tools for imaging specific targets seem poised for
significant advances in the coming months as well.

Closing the gap

In 1873, Ernest Abbe recognized that the resolving power of light microscopy
was limited such that two points could only be distinguished if they were
separated by more than half of the wavelength of light. This barrier was
overcome when optical laws were applied to electrons and the electron
microscope (EM) was created, arguably the most important advance in
microscopy since its introduction. With wavelengths 100,000 times smaller
than light, electron microscopes allowed for picometer resolution. Rather
than replacing light microscopy, EM has become its valuable companion. Light
microscopy maintains several advantages, such as the ability to image live
cells and amenability to 3D reconstruction of images that keeps this
technology at the front of engineers' and biologists' minds. In fact,
significant efforts are now focused on closing the resolution gap between
these types of microscopy.

Since Stefan Hell first overcame Abbe’s barrier by introducing the stimulated
emission depletion (STED) microscope in 2000, with which he obtained
fluorescent images with nanometer resolution, many other super-resolution
microscopes based on differing strategies have been developed and refined.
Initially, these microscopes were confined to specialized laboratories
requiring trained microscopists, but recent years have brought us a little
closer to readily accessible instruments that can be used by
non-specialists. In 2012 for example, Liu et al. modified Continuous Wave
STED, a variation of STED that does not require precise synchronization
between the pulsed excitation and STED lasers. They used a Ti:Sapphire
oscillator, which is more accessible than the lasers commonly required for
STED, and optimized the depletion wavelength with different dyes, and were
able to achieve super-resolution of 71nm, sufficient to image cytoskeletal
filaments and to visualize individual RNA granules of DYlight
650-MTRIP-labeled viral genomic RNA of human respiratory syncytial virus in
Hep2 cells for the first time (1).

Alternative approaches for super-resolution microscopy based on molecule
localization schemes also saw notable achievements in 2012. Some of these
include a solution for handling data from photoactivated localization
microscopy (PALM) using statistical algorithms to quantitatively describe
the spatial organization of molecules (2) and compressed sensing for images
with overlapping fluorescent spots, which allows for a density of activated
fluorophores an order of magnitude higher than previously accomplished (3).
As more super-resolution imaging tools are refined and their ease of use
enhanced, we expect new users to dip into the super-resolution imaging
waters in the coming months, resulting in new molecular insights and new
challenges for developers that will serve to further push super-resolution
imaging to the forefront of cell biology during 2013.

Clearing the way

While there are numerous technologies available now for imaging cellular
processes (confocal microscopes, super-resolution systems, total internal
reflectance, etc.), these did not develop in a vacuum, but side-by-side with
methods for preparing the samples to be examined. It's hard to say which
side has provided the bigger driving force since specimen preparation
improves to exploit the full potential of the microscope while microscopes
evolve to broaden and deepen what samples can be examined. Because these two
sides of microscopy are so intertwined, we can hardly predict advances in
technology without also expecting new approaches to prepare those cells,
organs, and organisms to be photographed.

Imaging a cell in its native context and environment is ideal for
understanding cellular function. Atsushi Miyawaki and his colleagues
recently brought us one step closer to this goal by introducing a urea-based
reagent, Scale, that can render a biological sample transparent,
thus avoiding light scattering that prevents imaging deep within tissues,
without affecting fluorescent signals. In mouse brains prepared with Scale,
fluorescently labeled neurons were imaged at subcellular resolution in situ
at a depth of several millimeters, permitting 3D reconstructions of neural
projections (4).

Of course, this approach depends on fluorescent protein labeling of the cells
under investigation as well. The array of proteins available for fluorescent
labeling has increased significantly since the 1990s when GFP was introduced
and continues to expand today. 2012 saw the development of cysteine-free
fluorescent proteins that maintain their proper conformations when expressed
in the endoplasmic reticulum (5), a new cyan fluorescent protein (CFP) with
the highest quantum yield yet reported for a monomeric fluorescent protein
and which was optimized following structural analysis of currently available
CFP, and other reports on the development of far red fluorescent proteins,
photoswitchable proteins, and a variety of new applications.

There is no question that 2013 will bring many more advances in microscopy and
we are eager to see each and every one. But we are even more excited to see
those unknown, unpredictable advances that are sure to come. Will we see a
new super-resolution approach or an reagent akin to Scale come out
in 2013? Only time will tell.